Almaas Runar, Pytte Morten, Lindstad Julie K, Wright Marianne, Saugstad Ola Didrik, Pleasure David, Rootwelt Terje
Department of Pediatric Research, Rikshospitalet, N-0027 Oslo, Norway.
J Neurochem. 2003 Mar;84(5):1018-27. doi: 10.1046/j.1471-4159.2003.01593.x.
To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post-mitotic NT2-N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP-ribose)-polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 +/- 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 +/- 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2-N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.
为研究细胞外酸中毒对缺血及复氧过程中细胞凋亡和坏死的影响，我们将人有丝分裂后NT2 - N神经元暴露于氧糖剥夺（OGD）后再进行复氧。在一些实验中，在OGD期间、复氧期间或两者期间将细胞培养基的pH值降至5.9。星形孢菌素用作细胞凋亡的阳性对照，可导致聚（ADP - 核糖）聚合酶（PARP）裂解和核碎裂，但OGD后未见PARP裂解且仅有少量碎裂。星形孢菌素处理后发现有低分子量DNA片段，但OGD后未发现。在OGD 3小时和复氧21小时后未观察到半胱天冬酶抑制剂的保护作用，但在复氧45小时后，半胱天冬酶抑制使3 -（4,5 - 二甲基噻唑 - 2 - 基）-2,5 - 二苯基四氮唑溴盐（MTT）裂解有适度改善。虽然OGD期间酸中毒并在复氧期间采用中性培养基可保护神经元（MTT：为中性培养基的228±117%，p < 0.001），但仅在复氧期间酸中毒则有害（MTT：38±25%，p < 0.01）。我们得出结论，在OGD后，凋亡机制在NT2 - N神经元中起次要作用。酸中毒对神经元存活的影响取决于酸中毒的时间，因为酸中毒在OGD期间具有保护作用，而在复氧期间则有害。
